ORIGINAL RESEARCH published: 14 June 2017 doi: 10.3389/fncel.2017.00168
Associative Memory Extinction Is Accompanied by Decayed Plasticity at Motor Cortical Neurons and Persistent Plasticity at Sensory Cortical Neurons Rui Guo 1† , Rongjing Ge 1† , Shidi Zhao 1† , Yulong Liu 1 , Xin Zhao 1 , Li Huang 1 , Sodong Guan 1 , Wei Lu 2 , Shan Cui 3 , Shirlene Wang 4 and Jin-Hui Wang 1,2,3,5 * 1
Department of Pathophysiology, Bengbu Medical College, Anhui, China, 2 School of Pharmacy, Qingdao University, Qingdao, China, 3 Brain and Cognitive Sciences, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China, 4 Department of Psychiatry and Behavioral Sciences, Feinberg School of Medicine, Northwestern University, Chicago, IL, United States, 5 Department of Biology, University of Chinese Academy of Sciences, Beijing, China
Edited by: Lavinia Alberi, University of Fribourg, Switzerland Reviewed by: Claire Cheetham, Carnegie Mellon University, United States Jürg Streit, University of Bern, Switzerland *Correspondence: Jin-Hui Wang [email protected] †
These authors have contributed equally to this work.
Received: 23 March 2017 Accepted: 31 May 2017 Published: 14 June 2017 Citation: Guo R, Ge R, Zhao S, Liu Y, Zhao X, Huang L, Guan S, Lu W, Cui S, Wang S and Wang J-H (2017) Associative Memory Extinction Is Accompanied by Decayed Plasticity at Motor Cortical Neurons and Persistent Plasticity at Sensory Cortical Neurons. Front. Cell. Neurosci. 11:168. doi: 10.3389/fncel.2017.00168
Associative memory is essential for cognition, in which associative memory cells and their plasticity presumably play important roles. The mechanism underlying associative memory extinction vs. maintenance remains unclear, which we have studied in a mouse model of cross-modal associative learning. Paired whisker and olfaction stimulations lead to a full establishment of odorant-induced whisker motion in training day 10, which almost disappears if paired stimulations are not given in a week, and then recovers after paired stimulation for an additional day. In mice that show associative memory, extinction and recovery, we have analyzed the dynamical plasticity of glutamatergic neurons in layers II–III of the barrel cortex and layers IV–V of the motor cortex. Compared with control mice, the rate of evoked spikes as well as the amplitude and frequency of excitatory postsynaptic currents increase, whereas the amplitude and frequency of inhibitory postsynaptic currents (IPSC) decrease at training day 10 in associative memory mice. Without paired training for a week, these plastic changes are persistent in the barrel cortex and decayed in the motor cortex. If paired training is given for an additional day to revoke associative memory, neuronal plasticity recovers in the motor cortex. Our study indicates persistent neuronal plasticity in the barrel cortex for crossmodal memory maintenance as well as the dynamical change of neuronal plasticity in the motor cortex for memory retrieval and extinction. In other words, the sensory cortices are essential for long-term memory while the behavior-related cortices with the inability of memory retrieval are correlated to memory extinction. Keywords: learning, memory, glutamate, GABA, neuron, synapse, barrel cortex and homeostasis
INTRODUCTION Associative learning is a common approach for information acquisition and associative memory is essential to logical reasoning and associative thinking (Wasserman and Miller, 1997; Suzuki, 2008; Wang and Cui, 2017). In terms of cellular mechanisms underlying associative learning and memory, associative memory cells are recruited in co-activated sensory cortices (Wang et al., 2015; Gao et al., 2016; Vincis and Fontanini, 2016; Yan et al., 2016) and their downstream
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plasticity was analyzed based on synaptic transmission and spiking capability by whole-cell recordings at glutamatergic neurons in the barrel and motor cortices, which were genetically labeled by yellow fluorescent protein (YFP) for the identification of these neurons (Feng et al., 2000) under the fluorescent microscope. Memory extinction stands for the loss of the stored information. Memory retrieval inability is termed as that the stored information is unable to be retrieved.
brain regions (Naya et al., 2003; Takehara-Nishiuchi and McNaughton, 2008; Viskontas, 2008; Cai et al., 2016), as well as use-dependent neural plasticity occurs during associative memory (Honey and Good, 2000; Blair et al., 2001; Christian and Thompson, 2003; Jones et al., 2003; Silva, 2003; Zhang et al., 2004; Dityatev and Bolshakov, 2005; Fanselow and Poulos, 2005; Weeks et al., 2007; Frey and Frey, 2008; Nikitin et al., 2008; Rosselet et al., 2011; Gao et al., 2016; Yan et al., 2016). On the other hand, memory extinction remains to be mechanistically elucidated, since it is necessary to know why memory losses occur during the mental retardation of neurological and psychological diseases, and how fear memory that leads to anxiety and major depression can be removed from the brains (Myers and Davis, 2002; Orsini and Maren, 2012; Baldi and Bucherelli, 2015; Giustino et al., 2016; Knox, 2016). If information storage is based on memory cells and their plasticity, the decreases in the level of neural plasticity and the number of memory cells may be associated with memory retrieval inability and even extinction. In the evaluation of memory extinction, the inability of information retrieval and recall through behavioral presentation is considered to be memory loss (Cammarota et al., 2005; Almeida-Corrêa and Amaral, 2014). Although the stored information cannot be recalled automatically and intentionally sometimes, its recall can be induced by the cues similar to or equal to the identities of primarily learned objects or events, indicating the information retention in the brain. This phenomenon suggests that the persistent storage of the learned information in certain brain areas and the attenuated ability to represent the stored information in behavior-related brain areas may be involved in memory extinction. It has been suggested that information retrievals triggered by the cues and presented by the behaviors are fulfilled by the neuronal circuits from sensory cortices to behavior-guide cortices through their relayed brain regions (Wang et al., 2015). This suggestion is granted by the facts that the stimulations to any of these areas can trigger memory retrievals (Ehrlich et al., 2009; Pape and Pare, 2010; Liu et al., 2012; Li et al., 2013; Xu and Südhof, 2013; Otis et al., 2017; Yokose et al., 2017) as well as the responses to associated signals can be recorded in sensory cortices (Wang et al., 2015; Vincis and Fontanini, 2016; Yan et al., 2016) and their downstream brain regions (Naya et al., 2003; Takehara-Nishiuchi and McNaughton, 2008; Viskontas, 2008; Cai et al., 2016). In other words, the sensory cortices are still primary locations for the signal storage and retrieval initiation (Wang et al., 2015; Gao et al., 2016). If this is a case, we should see the persistence of neuronal plasticity in the sensory cortices and the decay of neuronal plasticity in behavior-control cortices during memory extinction, as well as the recovery of neuronal plasticity in behavior-control cortices after memory restoration. To approach these questions above, we aimed to investigate cellular mechanisms underlying memory formation and retrieval inability in a mouse model of associative learning (Wang et al., 2015) by comparing neuronal plasticity at the sensory and motor cortices during the periods of associative memory establishment, extinction and reestablishment. Neuronal
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MATERIALS AND METHODS All experimental protocols were performed in accordance with the guidelines by the Administration Office of Laboratory Animals at Beijing China. All experiment protocols were approved by the Institutional Animal Care Unit Committee in the Administration Office of Laboratory Animals at Beijing China (B10831).
Mouse Model of Associative Memory To analyze cell-specific mechanism for associative memory we used C57 Thy1-YFP mice (Feng et al., 2000; Zhang et al., 2013), whose glutamatergic neurons were genetically labeled by YFP. Two groups of mice were trained at postnatal days 20 by the simultaneous pairing of mechanical whisker stimuli (WS) with odor stimuli (OS, butyl acetate toward the noses) and the unpairing of these stimuli (control), respectively (Wang et al., 2015; Gao et al., 2016; Yan et al., 2016). The stimulations for paired WS/OS and unpaired WS/OS mice were given by the multiple-sensory modal stimulator (MSMS, ZL201410499466), in which the intensities, time, frequency and intervals of OS and WS were precisely and consistently set. In unpairing group, the interval between WS and OS was randomly about 2–5 min. The OS intensity was sufficient to induce the response of olfactory bulb neurons seen by two-photon Ca2+ imaging, and the WS intensity was sufficient to evoke whisker fluctuation after WS ended. Each of these mice was trained 20 s in each time, five times per day with intervals of 2 h for consecutively 10 days. It is noteworthy that the intensities, time, frequency and total number of the WS and OS are same for paired and unpaired groups. During the training, each mouse was placed in a home-made cage. Care was taken to avoid stressful experimental condition and circadian disturbance to the mice that showed normal whisking and symmetric whiskers (Wang et al., 2015; Gao et al., 2016; Yan et al., 2016). Long whiskers (such as arcs 1–2) on the same side and rows were assigned for mechanical stimuli and for the observation of their responses to the odor-test. This selection was based on studies in cross-modal plasticity (Ni et al., 2010; Ye et al., 2012). We did not trim short whiskers since whisker trimming elevated the excitability of the barrel cortex (Zhang et al., 2013). Whisker motion tracks were monitored by a digital video camera (240 Hz) and were quantified in whisker retraction angle and whisking frequency (MB-Ruler, version 5.0 by Markus Bader, MB-Softwaresolution, Germany). Whisking angles were measured as angles lined from the original position to whisker
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Whole-Cell Recording
retraction. Whisking frequency was the times of whisker fluctuation per second (Hz). The response of mouse whiskers to the odor-test (butyl acetate, 20 s) was measured before the training and at the end of each training day to quantify the onset time and levels of conditioned reflex (CR). CR-formation was defined to meet the following criteria. The patterns of odorant-induced whisker motion were similar to those of WS-induced whisker motion. Whisking frequency and angles significantly increased, compared to those before the training. The approaches for statistical analyses are given in the section of statistical analysis. As this type of whisker motion induced by the odorant was originally induced by WS, the odor signal initiated a recall of the whisker signal and then led to whisker motion (Wang et al., 2015; Gao et al., 2016; Yan et al., 2016). The group of WS/OS-paired mice showing odorant-induced whisker motion was further divided into three subgroups for behavioral tests to show associative memory establishment, extinction and reestablishment as well as for electrophysiological study in the barrel and motor cortices to show dynamic changes in neuronal plasticity. That is, the mice show the establishment of odorant-induced whisker motion trained by WS/OS-pairing for 10 days, the mice show the decay of associative memory (odorant-induced whisker motion trained by the WS/OS pairing for 10 days) without further training in a subsequent week, and the mice show the reestablishment of associative memory by WS/OS-paired training for an additional day after the decay of odorant-induced whisker motion, as illustrated in Figure 1. These three groups of the mice were used for the studies of neuronal activities by electrophysiology.
Cortical neurons were recorded by MultiClamp-700B amplifier in voltage-clamp for their synaptic activities. The electrical signals were inputted into pClamp-10 (Axon Instrument Inc., CA, USA) for data acquisitions and analyses. The output bandwidth in this amplifier was 3 kHz. The pipette solution for studying excitatory synapses included (mM) 150 K-gluconate, 5 NaCl, 5 HEPES, 0.4 EGTA, 4 Mg-ATP, 0.5 Tris-GTP and 5 phosphocreatine (pH 7.35; (Ge et al., 2011, 2014)). The pipette solution for studying the inhibitory synapses contained (mM) 130 K-gluconate, 20 KCl, 5 NaCl, 5 HEPES, 0.5 EGTA, 4 Mg-ATP, 0.5 Tris–GTP and 5 phosphocreatine (Zhang et al., 2012). These pipette solutions were freshly made and filtered (0.1 µm), in which their osmolarity was 295–305 mOsmol and pipette resistance was 5–6 MΩ. The functions of the glutamatergic neurons were assessed based on their active intrinsic property, excitatory synaptic transmission and inhibitory synaptic transmission (Chen et al., 2008; Wang et al., 2008). The excitatory synaptic transmission was evaluated by recording spontaneous excitatory postsynaptic currents (sEPSCs) under the voltage-clamp on these glutamatergic neurons in presence of 10 µM bicuculline in ACSF to block ionotropic GABA receptors (Wang, 2003). Ten micro molar CNQX and 40 µM DAP-5 were added into ACSF perfused onto the slices at the end of experiments to examine whether the synaptic responses were mediated by GluRs, which blocked EPSCs in our experiments. Series and input resistances for all neurons were monitored by injecting hyperpolarization pulses (5 mV/50 ms), and calculated by voltage pulses vs. instantaneous and steady-state currents. Inhibitory synaptic transmission was evaluated by recording spontaneous inhibitory postsynaptic currents (sIPSCs) under the voltage-clamp on glutamatergic neurons in the presence of 10 µM 6-Cyano-7-nitroquinoxaline-2,3-(1H,4H)-dione (CNQX) and 40 µM D-amino-5-phosphonovanolenic acid (D-AP5) in ACSF to block ionotropic glutamate receptors (Wei et al., 2004; Ma et al., 2016a). Ten micro molar bicuculline was washed onto the slices at the end of experiments to examine whether synaptic responses were mediated by GABAA R, which blocked sIPSCs in our experiments. Series and input resistances in all of the neurons were monitored by injecting hyperpolarization pulses (5 mV/50 ms), and calculated by voltage pulses vs. instantaneous and steady-state currents. The pipette solution with the high concentration of chloride ions makes the reversal potential to be −42 mV. sIPSCs will be inward when the membrane holding potential at −65 (Wei et al., 2004; Wang G. Y. et al., 2016; Xu et al., 2016). Action potentials at these cortical neurons were induced by injecting depolarization pulses, whose intensity and duration were altered based on the aim of the experiments. The ability to convert excitatory inputs into digital spikes was evaluated by input-output (spikes per second vs. normalized stimuli) when various stimuli were given (Chen et al., 2006a,b, 2008; Wang et al., 2008), in which stimulus intensities were
Brain Slices and Neurons Cortical slices (400 µm) were prepared from the mice of CR-formation, CR-extinction and CR-recovery within 24 h after the training was ended. They were anesthetized by inhaling isoflurane and decapitated by the guillotine. The slices were cut by Vibratome in the oxygenated (95%O2 /5%CO2 ) artificial cerebrospinal fluid (ACSF), in which the chemical concentrations (mM) were 124 NaCl, 3 KCl, 1.2 NaH2 PO4 , 26 NaHCO3 , 0.5 CaCl2 , 4 MgSO4 , 10 dextrose and 5 HEPES, pH 7.35 at 4◦ C. The slices were held in the oxygenated ACSF (124 NaCl, 3 KCl, 1.2 NaH2 PO4 , 26 NaHCO3 , 2.4 CaCl2 , 1.3 MgSO4 , 10 dextrose and 5 HEPES, pH 7.35) at 25◦ C for 2 h. The slices were transferred to submersion chamber (Warner RC26G) that was perfused with the oxygenated ACSF at 31◦ C for whole-cell recording (Wang and Kelly, 2001). Electrophysiological recordings on YFP-labeled glutamatergic neurons in layers II-III of the barrel cortices as well as layers IV–V of the motor cortices were conducted under a DIC-fluorescent microscope (Nikon FN-E600, Japan). The wavelength at 575 nm excited YFP. These glutamatergic neurons showed pyramidal shape and regular spikes with adaptations of spike amplitudes and frequencies (DeFelipe et al., 2013; Lu et al., 2014; Xu et al., 2016). The cerebral slices were the coronal sections including the barrels correspondent to the projection from long whiskers that were stimulated in pairing WS and OS training.
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FIGURE 1 | The establishment, extinction and reestablishment of odorant-induced whisker motion in mice. (A) Presents whisker motions in response to the odor-test (black traces on top) in conditioned reflex (CR)-formation mice and unpaired stimulus (UPS) mice at the days 1, 10, 17 and 18. The paired or unpaired trainings were given from day 1 to day 10 as well as at day 18, however, the non-training was between day 11 and 16. Calibration bars are 30◦ and 5 s. (B) illustrates the angles of whisker fluctuation in response to the odor-test during different training days in CR-formation mice (round symbols) and UPS mice (triangles), indicating odorant-induced whisker motion in terms of establishment, decay and reestablishment. (C) illustrates the comparisons of whisking angles in response to the odor-test in CR-formation mice (filled bars) and UPG mice (hollow bars) at training days 1, 10, 17 and 18. (D) shows the comparisons of whisking frequencies in response to the odor-test in CR-formation mice (filled bars) and UPG mice (hollow bars) at training days 1, 10, 17 and 18. (E) illustrates the comparisons of whisker retraction duration in response to the odor-test in CR-formation mice (filled bars) and UPG mice (hollow bars) at the training days 1, 10, 17 and 18. Two asterisks show p < 0.01 (Statistical significance was determined using two-way analysis of variance (ANOVA) with a Bonferroni correction for multiple comparisons).
Data were analyzed if the recorded neurons had resting membrane potentials negatively more than −70 mV and action potential amplitudes more than 100 mV. The criteria for the acceptance of each experiment also included less than 5% alternations in the resting membrane potential, spike magnitude, and input resistance throughout each experiment. Input resistance was monitored by measuring cellular responses to hyperpolarization pulse at the same values as the depolarization that evoked action potentials. In order to estimate the effects of associative learning on neuronal spikes and synaptic transmission, we measured the amplitudes and intervals of sEPSC and sIPSC as well as the input-output of spikes vs. stimulations under the conditions of control and associative memory including its establishment, extinction and reestablishment, which were presented as mean ± SE. sEPSC and sIPSC frequencies were calculated by 1/intervals. The comparisons of sEPSCs and sIPSCs among different groups were based on their values at 67% of cumulative probability.
step-increasing by 10% normalized stimulations. As the excitability of different neurons was variable, step-increased depolarization pulses were given based on their normalization. The base value of stimulus intensity for this normalization at each neuron was the threshold intensity of depolarization pulse (1000 ms in duration) to evoke a single spike (Chen et al., 2006b). We did not measure the rheobase to show neuronal excitability, since this strength-duration relationship was used to indicate the ability to fire a single spike. The recordings of spontaneous synaptic currents, instead of the evoked synaptic currents, are based on the following reasons. sEPSC and sIPSC amplitudes represent the responsiveness and the densities of the postsynaptic receptors. The frequencies imply the probability of transmitter release from an axon terminal and the number of the presynaptic axons innervated on the recorded neuron (Zucker and Regehr, 2002; Stevens, 2004). These parameters can be used to analyze presynaptic and postsynaptic mechanisms underlying neuronal plasticity, whereas the evoked postsynaptic currents cannot separate these mechanisms. We did not add TTX into the ACSF to record miniature postsynaptic currents since we had to record neuronal excitability (Ma et al., 2016b; Xu et al., 2016).
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Statistical Analyses The paired t-test was used in the comparisons of the experimental data before and after associative learning, before and after
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the barrel cortex (glutamatergic neurons in layers II–III) or the motor cortex (glutamatergic neurons in layers IV–V), sEPSC were recorded by whole-cell voltage-clamp to assess excitatory synaptic transmission. Input-output curves at these neurons were measured under current-clamp to evaluate their ability to convert excitatory inputs into spikes. sIPSCs were recorded to assess inhibitory synaptic function (Zhang et al., 2013; Gao et al., 2016).
bicuculine or CNQX/D-AP5 applications, as well as the neuronal responses to whisker stimulus and odorant stimulus in each of the mice. One-way analysis of variance (ANOVA) was applied to make the statistical comparisons in the changes of neuronal activities between control and CR-formation groups.
RESULTS
Persistent Maintenance of Spiking Ability at Barrel Cortical Neurons, but Not Motor Cortical Neurons
Dynamical Changes in Establishment, Extinction and Reestablishment of Odorant-Induced Whisker Motion
For studying the roles of glutamatergic neurons at barrel and motor cortices in memory maintenance and extinction, the mice that showed odorant-induced whisker motion were divided into three groups: CR-formation by WS/OS-paired training for 10 days (day 10), CR-formation by WS/OS-paired training for 10 days and then without WS/OS-pairing in a week for CR-extinction (day 17), as well as CR-recovery by WS/OS-paired training for an additional day (day 18). Their functional plasticity was compared with that in UPS mice. Sequential spikes on glutamatergic neurons from barrel and motor cortices were induced by depolarization pulse in CR-formation mice at training days 10, 17 and 18 as well as UPS mice (Figures 2A,B). Figure 2C shows spikes per second vs. normalized stimuli in barrel cortical neurons from CR-formation mice at training day 10 (red symbols; n = 11 cells from 6 mice), day 17 (blue; n = 12 cells from 6 mice) and day 18 (green; n = 10 cells from 7 mice), as well as those from UPS mice (cyan; n = 13 cells from 6 mice). Figure 2D shows spikes per second vs. normalized stimuli in motor cortical neurons from CR-formation mice at training day 10 (red symbols, n = 12), day 17 (blue, n = 12) and day 18 (green, n = 11), as well as those from UPS mice (cyan, n = 10). Spikes per second at the 3.0 of normalized stimuli in barrel cortical neurons from CR-formation mice are 16.64 ± 0.95 at day 10 (red bar in Figure 2E), 16.75 ± 0.98 at day 17 (blue) and 15.70 ± 0.56 at day 18 (green), compared to 12.92 ± 0.54 at those neurons from UPS mice (cyan). Spikes per second at the 3.0 of normalized stimuli in motor cortical neurons from CR-formation mice are 16 ± 0.86 at day 10 (red bar in Figure 2F), 11.58 ± 0.53 at day 17 (blue) and 15 ± 1.05 at day 18 (green), compared with 12.2 ± 0.61 at those from UPS mice (cyan). These results indicate that the increased spiking ability in barrel cortical glutamatergic neurons is maintained regardless of the extinction of crossmodal associative memory, while spiking ability in motor cortical glutamatergic neurons is decayed in the retrieval inability of associative memory.
Mice were trained by pairing WS and OS or WS/OS-unpaired stimulation (UPS; Wang et al., 2015; Gao et al., 2016; Yan et al., 2016). In WS/OS-paired mice, their whiskers respond to the odor-test after paired trainings for 10 days, i.e., odorantinduced whisker motion or CR. This CR-formation disappears without WS/OS-paired training in a week, and can be reevoked by the WS/OS-paired training for additional day (Figure 1A). Figure 1B shows statistical data about this odorant-induced whisker motion that is fully establishment at training day 10, decays within 1 week and recovers by WS/OS-pair for an additional day (circle symbols in Figure 1B; n = 10), compared with UPS mice (triangles; n = 10). Figures 1C–E shows whisking angle, whisking frequency and whisker retraction duration in CR-formation mice (filled bars) and UPS mice (hollow bars) at days 1, 10, 17 and 18. Whisking angles are 3.37 ± 0.22◦ at day 1, 12.20 ± 0.50◦ at day 10, 3.32 ± 0.43◦ at day 17 and 11.98 ± 0.54◦ at day 18 (Figure 1C). Whisking frequencies are 2.45 ± 0.37 Hz at day 1, 11.18 ± 0.54 Hz at day 10, 2.45 ± 0.37 Hz at day 17 and 10.75 ± 0.4 Hz at day 18 (Figure 1D). Whisker retraction durations are 1.25 ± 0.2 s at day 1, 3.20 ± 0.20 s at day 10, 1.31 ± 0.20 s at day 17 and 3.19 ± 0.2 s at day 18 (Figure 1E; two asterisks denote p < 0.01; One-way ANOVA). Therefore, odorant-induced whisker motion shows a full establishment by the WS/OSpairing for 10 days, the extinction without the WS/OS-pairing for 1 week and the reestablishment by the WS/OS-pairing for an additional day. In terms of cellular mechanisms underlying the establishment, extinction and reestablishment of cross-modal associative memory in CR-formation mice, we hypothesize that neuronal plasticity in cerebral cortices establishes, decays and reestablishes. As odorant-induced whisker motion is accompanied by the upregulations of glutamatergic neurons and synapses in the barrel cortices (Wang et al., 2015; Gao et al., 2016; Yan et al., 2016), we aim to study whether the upregulation, decay and re-upregulation of neuronal and synaptic activity in the barrel cortex and the motor cortex are parallel to and even correlated to the establishment, extinction and reestablishment of cross-modal associative memory. The analyses of neural plasticity at glutamatergic neurons included the functional changes in their ability to convert analog synaptic signals into digital spikes as well as their receptions to excitatory and inhibitory synaptic inputs in CR-formation mice and UPS mice. In the coronal directions of brain slices including
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Excitatory Synapse Upregulation and Inhibitory Synapse Downregulation Maintain at Barrel Cortical Neurons As shown in Figures 3A–D, spontaneous excitatory synaptic currents were recorded by whole-cell voltage-clamp on barrel
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from 6 mice). The insert in Figure 3E illustrates that sEPSC intervals at 67% of cumulative probability in these neurons from CR-formation mice are 158 ± 11 ms at day 10 (red bar), 138 ± 14 ms at day 17 (blue) and 174 ± 15 ms at day 18 (green), in comparison with 494 ± 36 ms from UPS mice (cyan; two asterisks, p < 0.01). Moreover, Figure 3F shows cumulative probability vs. sEPSC amplitudes on the glutamatergic neurons from CR-formation mice in training days 10 (red symbols), 17 (blue) and 18 (green) as well as those from UPS mice (cyan). The insert in Figure 3F shows that sEPSC amplitudes at 67% of cumulative probability in these neurons from CR-formation mice are 20.0 ± 0.80 pA at day 10 (red bar), 20.60 ± 1.30 pA at day 17 (blue) and 18.30 ± 1.10 pA at day 18 (green), compared with 10.40 ± 0.7 pA from UPS mice (cyan; two asterisks, p < 0.01). Therefore, the increase of excitatory synaptic transmission on barrel cortical glutamatergic neurons is maintained regardless of the retrieval inability of cross-modal associative memory. As shown in Figures 4A–D, spontaneous inhibitory synaptic currents were recorded by whole-cell voltage-clamp on barrel cortical glutamatergic neurons in CR-formation mice and UPS mice. Compared to UPS mice (cyan), sIPSC amplitude and frequency appear decreased in training days 10 (red trace), 17 (blue) and 18 (green). Figure 4E shows cumulative probability vs. sIPSC intervals on the glutamatergic neurons from CR-formation mice in training days 10 (red symbols; n = 11 cells from 6 mice), 17 (blue; n = 11 cells from 6 mice) and 18 (green; n = 9 cells from 6 mice) as well as those from UPS mice (cyan; n = 9 cells from 6 mice). The insert in Figure 4E shows that sIPSC intervals at 67% of cumulative probability in these neurons from CR-formation mice are 632 ± 22 ms at day 10 (red bar), 670 ± 31 ms at day 17 (blue) and 604 ± 38 ms at day 18 (green), in comparison with 281 ± 19 ms from UPS mice (cyan; two asterisks, p < 0.01). Figure 4F shows cumulative probability vs. sIPSC amplitudes on glutamatergic neurons from CR-formation mice in training days 10 (red symbols), 17 (blue) and 18 (green) as well as those in UPS mice (cyan). The insert in Figure 4F shows that sIPSC amplitudes at 67% of cumulative probability in these neurons from CR-formation mice are 10.3 ± 0.7 pA at day 10 (red bar), 9.2 ± 0.3 pA at day 17 (blue) and 10.4 ± 0.5 pA at day 18 (green), compared to 20.8 ± 1.4 pA from UPS mice (cyan; two asterisks, p < 0.01). Therefore, the decrease of inhibitory synaptic transmission on barrel cortical glutamatergic neurons is maintained regardless of the retrieval inability of cross-modal associative memory.
FIGURE 2 | The enhanced ability to encode spikes is maintained at barrel cortical glutamatergic neurons, but not at motor cortical glutamatergic neurons. In the UPS mice and CR-formation mice at training days 10, 17 and 18, the sequential spikes were induced by depolarization pulses under the current-clamp recording on the yellow fluorescent protein (YFP)-labeled glutamatergic neurons in brain slices. (A) illustrates the spikes induced by a depolarization pulse in barrel cortical glutamatergic neurons from UPS mouse (cyan trace) and from CR-formation mice at training days 10 (red), 17 (blue) and 18 (green). (B) illustrates the spikes induced by a depolarization pulse in motor cortical glutamatergic neurons from UPS mouse (cyan trace) and from CR-formation mice at training days 10 (red), 17 (blue) and 18 (green). (C) illustrates spikes per second vs. normalized stimuli in the barrel cortical neurons from UPS mouse (cyan symbols) and from CR-formation mice at training days 10 (red), 17 (blue) and 18 (green). (D) shows spikes per second vs. normalized stimuli in the motor cortical neurons from UPS mouse (cyan symbols) and from CR-formation mice at training days 10 (red), 17 (blue) and 18 (green). (E) shows statistical comparisons of spikes per second at 3.0 normalized stimuli in the barrel cortical neurons from UPS mouse (cyan bar, 12.92 ± 0.54, n = 13) and from CR-formation mice at training days 10 (red, 16.64 ± 0.95, n = 11), 17 (blue, 16.75 ± 0.98, n = 12) and 18 (green, 15.70 ± 0.56, n = 10; from left: p = 0.004, p = 0.934, p = 0.388). (F) shows statistical comparisons of spikes per second at 3.0 normalized stimuli in motor cortical neurons from UPS mouse (cyan bar, 12.2 ± 0.61, n = 10) and from CR-formation mice at training days 10 (red, 16 ± 0.86, n = 12), 17 (blue, 11.58 ± 0.53, n = 12) and 18 (green, 15 ± 1.05, n = 11; from left: p = 0.002, p =
Associative Memory Extinction Is Accompanied by Decayed Plasticity at Motor Cortical Neurons and Persistent Plasticity at Sensory Cortical Neurons Rui Guo 1† , Rongjing Ge 1† , Shidi Zhao 1† , Yulong Liu 1 , Xin Zhao 1 , Li Huang 1 , Sodong Guan 1 , Wei Lu 2 , Shan Cui 3 , Shirlene Wang 4 and Jin-Hui Wang 1,2,3,5 * 1
Department of Pathophysiology, Bengbu Medical College, Anhui, China, 2 School of Pharmacy, Qingdao University, Qingdao, China, 3 Brain and Cognitive Sciences, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China, 4 Department of Psychiatry and Behavioral Sciences, Feinberg School of Medicine, Northwestern University, Chicago, IL, United States, 5 Department of Biology, University of Chinese Academy of Sciences, Beijing, China
Edited by: Lavinia Alberi, University of Fribourg, Switzerland Reviewed by: Claire Cheetham, Carnegie Mellon University, United States Jürg Streit, University of Bern, Switzerland *Correspondence: Jin-Hui Wang [email protected] †
These authors have contributed equally to this work.
Received: 23 March 2017 Accepted: 31 May 2017 Published: 14 June 2017 Citation: Guo R, Ge R, Zhao S, Liu Y, Zhao X, Huang L, Guan S, Lu W, Cui S, Wang S and Wang J-H (2017) Associative Memory Extinction Is Accompanied by Decayed Plasticity at Motor Cortical Neurons and Persistent Plasticity at Sensory Cortical Neurons. Front. Cell. Neurosci. 11:168. doi: 10.3389/fncel.2017.00168
Associative memory is essential for cognition, in which associative memory cells and their plasticity presumably play important roles. The mechanism underlying associative memory extinction vs. maintenance remains unclear, which we have studied in a mouse model of cross-modal associative learning. Paired whisker and olfaction stimulations lead to a full establishment of odorant-induced whisker motion in training day 10, which almost disappears if paired stimulations are not given in a week, and then recovers after paired stimulation for an additional day. In mice that show associative memory, extinction and recovery, we have analyzed the dynamical plasticity of glutamatergic neurons in layers II–III of the barrel cortex and layers IV–V of the motor cortex. Compared with control mice, the rate of evoked spikes as well as the amplitude and frequency of excitatory postsynaptic currents increase, whereas the amplitude and frequency of inhibitory postsynaptic currents (IPSC) decrease at training day 10 in associative memory mice. Without paired training for a week, these plastic changes are persistent in the barrel cortex and decayed in the motor cortex. If paired training is given for an additional day to revoke associative memory, neuronal plasticity recovers in the motor cortex. Our study indicates persistent neuronal plasticity in the barrel cortex for crossmodal memory maintenance as well as the dynamical change of neuronal plasticity in the motor cortex for memory retrieval and extinction. In other words, the sensory cortices are essential for long-term memory while the behavior-related cortices with the inability of memory retrieval are correlated to memory extinction. Keywords: learning, memory, glutamate, GABA, neuron, synapse, barrel cortex and homeostasis
INTRODUCTION Associative learning is a common approach for information acquisition and associative memory is essential to logical reasoning and associative thinking (Wasserman and Miller, 1997; Suzuki, 2008; Wang and Cui, 2017). In terms of cellular mechanisms underlying associative learning and memory, associative memory cells are recruited in co-activated sensory cortices (Wang et al., 2015; Gao et al., 2016; Vincis and Fontanini, 2016; Yan et al., 2016) and their downstream
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plasticity was analyzed based on synaptic transmission and spiking capability by whole-cell recordings at glutamatergic neurons in the barrel and motor cortices, which were genetically labeled by yellow fluorescent protein (YFP) for the identification of these neurons (Feng et al., 2000) under the fluorescent microscope. Memory extinction stands for the loss of the stored information. Memory retrieval inability is termed as that the stored information is unable to be retrieved.
brain regions (Naya et al., 2003; Takehara-Nishiuchi and McNaughton, 2008; Viskontas, 2008; Cai et al., 2016), as well as use-dependent neural plasticity occurs during associative memory (Honey and Good, 2000; Blair et al., 2001; Christian and Thompson, 2003; Jones et al., 2003; Silva, 2003; Zhang et al., 2004; Dityatev and Bolshakov, 2005; Fanselow and Poulos, 2005; Weeks et al., 2007; Frey and Frey, 2008; Nikitin et al., 2008; Rosselet et al., 2011; Gao et al., 2016; Yan et al., 2016). On the other hand, memory extinction remains to be mechanistically elucidated, since it is necessary to know why memory losses occur during the mental retardation of neurological and psychological diseases, and how fear memory that leads to anxiety and major depression can be removed from the brains (Myers and Davis, 2002; Orsini and Maren, 2012; Baldi and Bucherelli, 2015; Giustino et al., 2016; Knox, 2016). If information storage is based on memory cells and their plasticity, the decreases in the level of neural plasticity and the number of memory cells may be associated with memory retrieval inability and even extinction. In the evaluation of memory extinction, the inability of information retrieval and recall through behavioral presentation is considered to be memory loss (Cammarota et al., 2005; Almeida-Corrêa and Amaral, 2014). Although the stored information cannot be recalled automatically and intentionally sometimes, its recall can be induced by the cues similar to or equal to the identities of primarily learned objects or events, indicating the information retention in the brain. This phenomenon suggests that the persistent storage of the learned information in certain brain areas and the attenuated ability to represent the stored information in behavior-related brain areas may be involved in memory extinction. It has been suggested that information retrievals triggered by the cues and presented by the behaviors are fulfilled by the neuronal circuits from sensory cortices to behavior-guide cortices through their relayed brain regions (Wang et al., 2015). This suggestion is granted by the facts that the stimulations to any of these areas can trigger memory retrievals (Ehrlich et al., 2009; Pape and Pare, 2010; Liu et al., 2012; Li et al., 2013; Xu and Südhof, 2013; Otis et al., 2017; Yokose et al., 2017) as well as the responses to associated signals can be recorded in sensory cortices (Wang et al., 2015; Vincis and Fontanini, 2016; Yan et al., 2016) and their downstream brain regions (Naya et al., 2003; Takehara-Nishiuchi and McNaughton, 2008; Viskontas, 2008; Cai et al., 2016). In other words, the sensory cortices are still primary locations for the signal storage and retrieval initiation (Wang et al., 2015; Gao et al., 2016). If this is a case, we should see the persistence of neuronal plasticity in the sensory cortices and the decay of neuronal plasticity in behavior-control cortices during memory extinction, as well as the recovery of neuronal plasticity in behavior-control cortices after memory restoration. To approach these questions above, we aimed to investigate cellular mechanisms underlying memory formation and retrieval inability in a mouse model of associative learning (Wang et al., 2015) by comparing neuronal plasticity at the sensory and motor cortices during the periods of associative memory establishment, extinction and reestablishment. Neuronal
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MATERIALS AND METHODS All experimental protocols were performed in accordance with the guidelines by the Administration Office of Laboratory Animals at Beijing China. All experiment protocols were approved by the Institutional Animal Care Unit Committee in the Administration Office of Laboratory Animals at Beijing China (B10831).
Mouse Model of Associative Memory To analyze cell-specific mechanism for associative memory we used C57 Thy1-YFP mice (Feng et al., 2000; Zhang et al., 2013), whose glutamatergic neurons were genetically labeled by YFP. Two groups of mice were trained at postnatal days 20 by the simultaneous pairing of mechanical whisker stimuli (WS) with odor stimuli (OS, butyl acetate toward the noses) and the unpairing of these stimuli (control), respectively (Wang et al., 2015; Gao et al., 2016; Yan et al., 2016). The stimulations for paired WS/OS and unpaired WS/OS mice were given by the multiple-sensory modal stimulator (MSMS, ZL201410499466), in which the intensities, time, frequency and intervals of OS and WS were precisely and consistently set. In unpairing group, the interval between WS and OS was randomly about 2–5 min. The OS intensity was sufficient to induce the response of olfactory bulb neurons seen by two-photon Ca2+ imaging, and the WS intensity was sufficient to evoke whisker fluctuation after WS ended. Each of these mice was trained 20 s in each time, five times per day with intervals of 2 h for consecutively 10 days. It is noteworthy that the intensities, time, frequency and total number of the WS and OS are same for paired and unpaired groups. During the training, each mouse was placed in a home-made cage. Care was taken to avoid stressful experimental condition and circadian disturbance to the mice that showed normal whisking and symmetric whiskers (Wang et al., 2015; Gao et al., 2016; Yan et al., 2016). Long whiskers (such as arcs 1–2) on the same side and rows were assigned for mechanical stimuli and for the observation of their responses to the odor-test. This selection was based on studies in cross-modal plasticity (Ni et al., 2010; Ye et al., 2012). We did not trim short whiskers since whisker trimming elevated the excitability of the barrel cortex (Zhang et al., 2013). Whisker motion tracks were monitored by a digital video camera (240 Hz) and were quantified in whisker retraction angle and whisking frequency (MB-Ruler, version 5.0 by Markus Bader, MB-Softwaresolution, Germany). Whisking angles were measured as angles lined from the original position to whisker
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Whole-Cell Recording
retraction. Whisking frequency was the times of whisker fluctuation per second (Hz). The response of mouse whiskers to the odor-test (butyl acetate, 20 s) was measured before the training and at the end of each training day to quantify the onset time and levels of conditioned reflex (CR). CR-formation was defined to meet the following criteria. The patterns of odorant-induced whisker motion were similar to those of WS-induced whisker motion. Whisking frequency and angles significantly increased, compared to those before the training. The approaches for statistical analyses are given in the section of statistical analysis. As this type of whisker motion induced by the odorant was originally induced by WS, the odor signal initiated a recall of the whisker signal and then led to whisker motion (Wang et al., 2015; Gao et al., 2016; Yan et al., 2016). The group of WS/OS-paired mice showing odorant-induced whisker motion was further divided into three subgroups for behavioral tests to show associative memory establishment, extinction and reestablishment as well as for electrophysiological study in the barrel and motor cortices to show dynamic changes in neuronal plasticity. That is, the mice show the establishment of odorant-induced whisker motion trained by WS/OS-pairing for 10 days, the mice show the decay of associative memory (odorant-induced whisker motion trained by the WS/OS pairing for 10 days) without further training in a subsequent week, and the mice show the reestablishment of associative memory by WS/OS-paired training for an additional day after the decay of odorant-induced whisker motion, as illustrated in Figure 1. These three groups of the mice were used for the studies of neuronal activities by electrophysiology.
Cortical neurons were recorded by MultiClamp-700B amplifier in voltage-clamp for their synaptic activities. The electrical signals were inputted into pClamp-10 (Axon Instrument Inc., CA, USA) for data acquisitions and analyses. The output bandwidth in this amplifier was 3 kHz. The pipette solution for studying excitatory synapses included (mM) 150 K-gluconate, 5 NaCl, 5 HEPES, 0.4 EGTA, 4 Mg-ATP, 0.5 Tris-GTP and 5 phosphocreatine (pH 7.35; (Ge et al., 2011, 2014)). The pipette solution for studying the inhibitory synapses contained (mM) 130 K-gluconate, 20 KCl, 5 NaCl, 5 HEPES, 0.5 EGTA, 4 Mg-ATP, 0.5 Tris–GTP and 5 phosphocreatine (Zhang et al., 2012). These pipette solutions were freshly made and filtered (0.1 µm), in which their osmolarity was 295–305 mOsmol and pipette resistance was 5–6 MΩ. The functions of the glutamatergic neurons were assessed based on their active intrinsic property, excitatory synaptic transmission and inhibitory synaptic transmission (Chen et al., 2008; Wang et al., 2008). The excitatory synaptic transmission was evaluated by recording spontaneous excitatory postsynaptic currents (sEPSCs) under the voltage-clamp on these glutamatergic neurons in presence of 10 µM bicuculline in ACSF to block ionotropic GABA receptors (Wang, 2003). Ten micro molar CNQX and 40 µM DAP-5 were added into ACSF perfused onto the slices at the end of experiments to examine whether the synaptic responses were mediated by GluRs, which blocked EPSCs in our experiments. Series and input resistances for all neurons were monitored by injecting hyperpolarization pulses (5 mV/50 ms), and calculated by voltage pulses vs. instantaneous and steady-state currents. Inhibitory synaptic transmission was evaluated by recording spontaneous inhibitory postsynaptic currents (sIPSCs) under the voltage-clamp on glutamatergic neurons in the presence of 10 µM 6-Cyano-7-nitroquinoxaline-2,3-(1H,4H)-dione (CNQX) and 40 µM D-amino-5-phosphonovanolenic acid (D-AP5) in ACSF to block ionotropic glutamate receptors (Wei et al., 2004; Ma et al., 2016a). Ten micro molar bicuculline was washed onto the slices at the end of experiments to examine whether synaptic responses were mediated by GABAA R, which blocked sIPSCs in our experiments. Series and input resistances in all of the neurons were monitored by injecting hyperpolarization pulses (5 mV/50 ms), and calculated by voltage pulses vs. instantaneous and steady-state currents. The pipette solution with the high concentration of chloride ions makes the reversal potential to be −42 mV. sIPSCs will be inward when the membrane holding potential at −65 (Wei et al., 2004; Wang G. Y. et al., 2016; Xu et al., 2016). Action potentials at these cortical neurons were induced by injecting depolarization pulses, whose intensity and duration were altered based on the aim of the experiments. The ability to convert excitatory inputs into digital spikes was evaluated by input-output (spikes per second vs. normalized stimuli) when various stimuli were given (Chen et al., 2006a,b, 2008; Wang et al., 2008), in which stimulus intensities were
Brain Slices and Neurons Cortical slices (400 µm) were prepared from the mice of CR-formation, CR-extinction and CR-recovery within 24 h after the training was ended. They were anesthetized by inhaling isoflurane and decapitated by the guillotine. The slices were cut by Vibratome in the oxygenated (95%O2 /5%CO2 ) artificial cerebrospinal fluid (ACSF), in which the chemical concentrations (mM) were 124 NaCl, 3 KCl, 1.2 NaH2 PO4 , 26 NaHCO3 , 0.5 CaCl2 , 4 MgSO4 , 10 dextrose and 5 HEPES, pH 7.35 at 4◦ C. The slices were held in the oxygenated ACSF (124 NaCl, 3 KCl, 1.2 NaH2 PO4 , 26 NaHCO3 , 2.4 CaCl2 , 1.3 MgSO4 , 10 dextrose and 5 HEPES, pH 7.35) at 25◦ C for 2 h. The slices were transferred to submersion chamber (Warner RC26G) that was perfused with the oxygenated ACSF at 31◦ C for whole-cell recording (Wang and Kelly, 2001). Electrophysiological recordings on YFP-labeled glutamatergic neurons in layers II-III of the barrel cortices as well as layers IV–V of the motor cortices were conducted under a DIC-fluorescent microscope (Nikon FN-E600, Japan). The wavelength at 575 nm excited YFP. These glutamatergic neurons showed pyramidal shape and regular spikes with adaptations of spike amplitudes and frequencies (DeFelipe et al., 2013; Lu et al., 2014; Xu et al., 2016). The cerebral slices were the coronal sections including the barrels correspondent to the projection from long whiskers that were stimulated in pairing WS and OS training.
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FIGURE 1 | The establishment, extinction and reestablishment of odorant-induced whisker motion in mice. (A) Presents whisker motions in response to the odor-test (black traces on top) in conditioned reflex (CR)-formation mice and unpaired stimulus (UPS) mice at the days 1, 10, 17 and 18. The paired or unpaired trainings were given from day 1 to day 10 as well as at day 18, however, the non-training was between day 11 and 16. Calibration bars are 30◦ and 5 s. (B) illustrates the angles of whisker fluctuation in response to the odor-test during different training days in CR-formation mice (round symbols) and UPS mice (triangles), indicating odorant-induced whisker motion in terms of establishment, decay and reestablishment. (C) illustrates the comparisons of whisking angles in response to the odor-test in CR-formation mice (filled bars) and UPG mice (hollow bars) at training days 1, 10, 17 and 18. (D) shows the comparisons of whisking frequencies in response to the odor-test in CR-formation mice (filled bars) and UPG mice (hollow bars) at training days 1, 10, 17 and 18. (E) illustrates the comparisons of whisker retraction duration in response to the odor-test in CR-formation mice (filled bars) and UPG mice (hollow bars) at the training days 1, 10, 17 and 18. Two asterisks show p < 0.01 (Statistical significance was determined using two-way analysis of variance (ANOVA) with a Bonferroni correction for multiple comparisons).
Data were analyzed if the recorded neurons had resting membrane potentials negatively more than −70 mV and action potential amplitudes more than 100 mV. The criteria for the acceptance of each experiment also included less than 5% alternations in the resting membrane potential, spike magnitude, and input resistance throughout each experiment. Input resistance was monitored by measuring cellular responses to hyperpolarization pulse at the same values as the depolarization that evoked action potentials. In order to estimate the effects of associative learning on neuronal spikes and synaptic transmission, we measured the amplitudes and intervals of sEPSC and sIPSC as well as the input-output of spikes vs. stimulations under the conditions of control and associative memory including its establishment, extinction and reestablishment, which were presented as mean ± SE. sEPSC and sIPSC frequencies were calculated by 1/intervals. The comparisons of sEPSCs and sIPSCs among different groups were based on their values at 67% of cumulative probability.
step-increasing by 10% normalized stimulations. As the excitability of different neurons was variable, step-increased depolarization pulses were given based on their normalization. The base value of stimulus intensity for this normalization at each neuron was the threshold intensity of depolarization pulse (1000 ms in duration) to evoke a single spike (Chen et al., 2006b). We did not measure the rheobase to show neuronal excitability, since this strength-duration relationship was used to indicate the ability to fire a single spike. The recordings of spontaneous synaptic currents, instead of the evoked synaptic currents, are based on the following reasons. sEPSC and sIPSC amplitudes represent the responsiveness and the densities of the postsynaptic receptors. The frequencies imply the probability of transmitter release from an axon terminal and the number of the presynaptic axons innervated on the recorded neuron (Zucker and Regehr, 2002; Stevens, 2004). These parameters can be used to analyze presynaptic and postsynaptic mechanisms underlying neuronal plasticity, whereas the evoked postsynaptic currents cannot separate these mechanisms. We did not add TTX into the ACSF to record miniature postsynaptic currents since we had to record neuronal excitability (Ma et al., 2016b; Xu et al., 2016).
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Statistical Analyses The paired t-test was used in the comparisons of the experimental data before and after associative learning, before and after
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the barrel cortex (glutamatergic neurons in layers II–III) or the motor cortex (glutamatergic neurons in layers IV–V), sEPSC were recorded by whole-cell voltage-clamp to assess excitatory synaptic transmission. Input-output curves at these neurons were measured under current-clamp to evaluate their ability to convert excitatory inputs into spikes. sIPSCs were recorded to assess inhibitory synaptic function (Zhang et al., 2013; Gao et al., 2016).
bicuculine or CNQX/D-AP5 applications, as well as the neuronal responses to whisker stimulus and odorant stimulus in each of the mice. One-way analysis of variance (ANOVA) was applied to make the statistical comparisons in the changes of neuronal activities between control and CR-formation groups.
RESULTS
Persistent Maintenance of Spiking Ability at Barrel Cortical Neurons, but Not Motor Cortical Neurons
Dynamical Changes in Establishment, Extinction and Reestablishment of Odorant-Induced Whisker Motion
For studying the roles of glutamatergic neurons at barrel and motor cortices in memory maintenance and extinction, the mice that showed odorant-induced whisker motion were divided into three groups: CR-formation by WS/OS-paired training for 10 days (day 10), CR-formation by WS/OS-paired training for 10 days and then without WS/OS-pairing in a week for CR-extinction (day 17), as well as CR-recovery by WS/OS-paired training for an additional day (day 18). Their functional plasticity was compared with that in UPS mice. Sequential spikes on glutamatergic neurons from barrel and motor cortices were induced by depolarization pulse in CR-formation mice at training days 10, 17 and 18 as well as UPS mice (Figures 2A,B). Figure 2C shows spikes per second vs. normalized stimuli in barrel cortical neurons from CR-formation mice at training day 10 (red symbols; n = 11 cells from 6 mice), day 17 (blue; n = 12 cells from 6 mice) and day 18 (green; n = 10 cells from 7 mice), as well as those from UPS mice (cyan; n = 13 cells from 6 mice). Figure 2D shows spikes per second vs. normalized stimuli in motor cortical neurons from CR-formation mice at training day 10 (red symbols, n = 12), day 17 (blue, n = 12) and day 18 (green, n = 11), as well as those from UPS mice (cyan, n = 10). Spikes per second at the 3.0 of normalized stimuli in barrel cortical neurons from CR-formation mice are 16.64 ± 0.95 at day 10 (red bar in Figure 2E), 16.75 ± 0.98 at day 17 (blue) and 15.70 ± 0.56 at day 18 (green), compared to 12.92 ± 0.54 at those neurons from UPS mice (cyan). Spikes per second at the 3.0 of normalized stimuli in motor cortical neurons from CR-formation mice are 16 ± 0.86 at day 10 (red bar in Figure 2F), 11.58 ± 0.53 at day 17 (blue) and 15 ± 1.05 at day 18 (green), compared with 12.2 ± 0.61 at those from UPS mice (cyan). These results indicate that the increased spiking ability in barrel cortical glutamatergic neurons is maintained regardless of the extinction of crossmodal associative memory, while spiking ability in motor cortical glutamatergic neurons is decayed in the retrieval inability of associative memory.
Mice were trained by pairing WS and OS or WS/OS-unpaired stimulation (UPS; Wang et al., 2015; Gao et al., 2016; Yan et al., 2016). In WS/OS-paired mice, their whiskers respond to the odor-test after paired trainings for 10 days, i.e., odorantinduced whisker motion or CR. This CR-formation disappears without WS/OS-paired training in a week, and can be reevoked by the WS/OS-paired training for additional day (Figure 1A). Figure 1B shows statistical data about this odorant-induced whisker motion that is fully establishment at training day 10, decays within 1 week and recovers by WS/OS-pair for an additional day (circle symbols in Figure 1B; n = 10), compared with UPS mice (triangles; n = 10). Figures 1C–E shows whisking angle, whisking frequency and whisker retraction duration in CR-formation mice (filled bars) and UPS mice (hollow bars) at days 1, 10, 17 and 18. Whisking angles are 3.37 ± 0.22◦ at day 1, 12.20 ± 0.50◦ at day 10, 3.32 ± 0.43◦ at day 17 and 11.98 ± 0.54◦ at day 18 (Figure 1C). Whisking frequencies are 2.45 ± 0.37 Hz at day 1, 11.18 ± 0.54 Hz at day 10, 2.45 ± 0.37 Hz at day 17 and 10.75 ± 0.4 Hz at day 18 (Figure 1D). Whisker retraction durations are 1.25 ± 0.2 s at day 1, 3.20 ± 0.20 s at day 10, 1.31 ± 0.20 s at day 17 and 3.19 ± 0.2 s at day 18 (Figure 1E; two asterisks denote p < 0.01; One-way ANOVA). Therefore, odorant-induced whisker motion shows a full establishment by the WS/OSpairing for 10 days, the extinction without the WS/OS-pairing for 1 week and the reestablishment by the WS/OS-pairing for an additional day. In terms of cellular mechanisms underlying the establishment, extinction and reestablishment of cross-modal associative memory in CR-formation mice, we hypothesize that neuronal plasticity in cerebral cortices establishes, decays and reestablishes. As odorant-induced whisker motion is accompanied by the upregulations of glutamatergic neurons and synapses in the barrel cortices (Wang et al., 2015; Gao et al., 2016; Yan et al., 2016), we aim to study whether the upregulation, decay and re-upregulation of neuronal and synaptic activity in the barrel cortex and the motor cortex are parallel to and even correlated to the establishment, extinction and reestablishment of cross-modal associative memory. The analyses of neural plasticity at glutamatergic neurons included the functional changes in their ability to convert analog synaptic signals into digital spikes as well as their receptions to excitatory and inhibitory synaptic inputs in CR-formation mice and UPS mice. In the coronal directions of brain slices including
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Excitatory Synapse Upregulation and Inhibitory Synapse Downregulation Maintain at Barrel Cortical Neurons As shown in Figures 3A–D, spontaneous excitatory synaptic currents were recorded by whole-cell voltage-clamp on barrel
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from 6 mice). The insert in Figure 3E illustrates that sEPSC intervals at 67% of cumulative probability in these neurons from CR-formation mice are 158 ± 11 ms at day 10 (red bar), 138 ± 14 ms at day 17 (blue) and 174 ± 15 ms at day 18 (green), in comparison with 494 ± 36 ms from UPS mice (cyan; two asterisks, p < 0.01). Moreover, Figure 3F shows cumulative probability vs. sEPSC amplitudes on the glutamatergic neurons from CR-formation mice in training days 10 (red symbols), 17 (blue) and 18 (green) as well as those from UPS mice (cyan). The insert in Figure 3F shows that sEPSC amplitudes at 67% of cumulative probability in these neurons from CR-formation mice are 20.0 ± 0.80 pA at day 10 (red bar), 20.60 ± 1.30 pA at day 17 (blue) and 18.30 ± 1.10 pA at day 18 (green), compared with 10.40 ± 0.7 pA from UPS mice (cyan; two asterisks, p < 0.01). Therefore, the increase of excitatory synaptic transmission on barrel cortical glutamatergic neurons is maintained regardless of the retrieval inability of cross-modal associative memory. As shown in Figures 4A–D, spontaneous inhibitory synaptic currents were recorded by whole-cell voltage-clamp on barrel cortical glutamatergic neurons in CR-formation mice and UPS mice. Compared to UPS mice (cyan), sIPSC amplitude and frequency appear decreased in training days 10 (red trace), 17 (blue) and 18 (green). Figure 4E shows cumulative probability vs. sIPSC intervals on the glutamatergic neurons from CR-formation mice in training days 10 (red symbols; n = 11 cells from 6 mice), 17 (blue; n = 11 cells from 6 mice) and 18 (green; n = 9 cells from 6 mice) as well as those from UPS mice (cyan; n = 9 cells from 6 mice). The insert in Figure 4E shows that sIPSC intervals at 67% of cumulative probability in these neurons from CR-formation mice are 632 ± 22 ms at day 10 (red bar), 670 ± 31 ms at day 17 (blue) and 604 ± 38 ms at day 18 (green), in comparison with 281 ± 19 ms from UPS mice (cyan; two asterisks, p < 0.01). Figure 4F shows cumulative probability vs. sIPSC amplitudes on glutamatergic neurons from CR-formation mice in training days 10 (red symbols), 17 (blue) and 18 (green) as well as those in UPS mice (cyan). The insert in Figure 4F shows that sIPSC amplitudes at 67% of cumulative probability in these neurons from CR-formation mice are 10.3 ± 0.7 pA at day 10 (red bar), 9.2 ± 0.3 pA at day 17 (blue) and 10.4 ± 0.5 pA at day 18 (green), compared to 20.8 ± 1.4 pA from UPS mice (cyan; two asterisks, p < 0.01). Therefore, the decrease of inhibitory synaptic transmission on barrel cortical glutamatergic neurons is maintained regardless of the retrieval inability of cross-modal associative memory.
FIGURE 2 | The enhanced ability to encode spikes is maintained at barrel cortical glutamatergic neurons, but not at motor cortical glutamatergic neurons. In the UPS mice and CR-formation mice at training days 10, 17 and 18, the sequential spikes were induced by depolarization pulses under the current-clamp recording on the yellow fluorescent protein (YFP)-labeled glutamatergic neurons in brain slices. (A) illustrates the spikes induced by a depolarization pulse in barrel cortical glutamatergic neurons from UPS mouse (cyan trace) and from CR-formation mice at training days 10 (red), 17 (blue) and 18 (green). (B) illustrates the spikes induced by a depolarization pulse in motor cortical glutamatergic neurons from UPS mouse (cyan trace) and from CR-formation mice at training days 10 (red), 17 (blue) and 18 (green). (C) illustrates spikes per second vs. normalized stimuli in the barrel cortical neurons from UPS mouse (cyan symbols) and from CR-formation mice at training days 10 (red), 17 (blue) and 18 (green). (D) shows spikes per second vs. normalized stimuli in the motor cortical neurons from UPS mouse (cyan symbols) and from CR-formation mice at training days 10 (red), 17 (blue) and 18 (green). (E) shows statistical comparisons of spikes per second at 3.0 normalized stimuli in the barrel cortical neurons from UPS mouse (cyan bar, 12.92 ± 0.54, n = 13) and from CR-formation mice at training days 10 (red, 16.64 ± 0.95, n = 11), 17 (blue, 16.75 ± 0.98, n = 12) and 18 (green, 15.70 ± 0.56, n = 10; from left: p = 0.004, p = 0.934, p = 0.388). (F) shows statistical comparisons of spikes per second at 3.0 normalized stimuli in motor cortical neurons from UPS mouse (cyan bar, 12.2 ± 0.61, n = 10) and from CR-formation mice at training days 10 (red, 16 ± 0.86, n = 12), 17 (blue, 11.58 ± 0.53, n = 12) and 18 (green, 15 ± 1.05, n = 11; from left: p = 0.002, p =
